Memory deficits in patients with AD is one of the most important factors affecting the quality of life. Some studies have found that the targeting of HDACI to class Ⅰ HDACs can improve the contextual memory and spatial memory deficits in the mouse model of AD[17, 18]. These results indicate that targeting class Ⅰ HDACs might be a promising approach for the intervention of AD. APP is a protein that has been widely studied in the field of AD pathology[19, 20]. APPsw-transfected neuron-like cells have robust expression of APP and are a reliable system of producing Aβ in vitro that can model the pathological characteristics of AD. SH-SY5Y cell was derived from human neuroblastoma, which could be induced by All-trans retinoic acid (RA) into the neuronal differentiation. In the study, the cells were transfected with a Recombinant Plasmid p-EGFP-N2-APPsw for establishing an Alzheimer's disease cell model. The increased expression of APP and Aβ and the increased level of tau phosphorylation indicated that the cell model was successfully constructed and could be used in subsequent in vitro experiments. Furthermore, the APP/Ps1 transgenic mouse was used as an animal AD model in vivo. We investigated the protective effects of BG45 on APPsw gene-transfected SH-SY5Y cells and APP/Ps1 transgenic mice. In our previous studies, we found that BG45 ameliorated the decrease in synaptic protein expression caused by exogenous Aβ by peripheral administration in cells and animal models. Therefore, to further explore the underlying mechanism of HDACI in improving synaptic plasticity, in this study, we used gene transfection to mediate the production of Aβ by cells themselves to study the effects of HDACI on early AD, and the relationship between the HDACI, synaptic protein, synapse-related genes and receptors.
It has been reported that the expression of HDAC2 is significantly increased in the brains of AD patients and the CK-p25 and 5xFAD transgenic mouse AD models and in in vitro AD-related neurotoxicity injury models. Overexpression of HDAC2 causes decreased dendritic spine density, synapse number, synaptic plasticity and memory formation, whereas overexpression of HDAC1 does not produce such effects. In addition, Lentivirus-mediated overexpression of HDAC3 used in the APP/Ps1mice also activated microglia cells, increased the level of Aβ and decreased the spine density. In this study, data showed that the expression of HDAC2, HDAC3 in APPsw cells were higher than in control cells.
To verify the correlation between HDACs and synapse-associated proteins in the AD cell model, we detected the expression of AD-related proteins and synapse-related proteins at 24 h, 36 h, and 48 h. The results showed that APP protein level was high in the APPsw group at 24 h. Moreover, we observed that spinophilin showed a significant decrease at 24 h; further, SYP and PSD-95 levels began to decrease at 36 h, which was consistent with the increase in HDAC1、HDAC2 and HDAC3. Kilgore, M., et al. reported that after inhibition of HDAC1, 2, and 3 by RGFP963, the result of contextual fear conditioning test in APP/PS1 mice showed that the inhibitors improved synaptogenesis and memory impairment. While lentivirus-mediated shRNA inhibition of HDAC3 reduced amyloid burden and Aβ levels, and rescued spatial memory impairment in APP/PS1 mice. Therefore, we assumed that, in the study, decreased expression of synapse-related proteins is associated with increased expression of HDACs. As a structural component of the presynaptic membrane and synaptic vesicles, SYP is a representative structural protein involved in the neurogenic budding reaction, and it is specifically distributed in the presynaptic membrane. It is mainly transported to the terminal ends of axons after neuron cell synthesis. The expression levels of SYP and PSD-95 can be used to assess synaptic distribution and density. We found that BG45 reduced the expression of HDAC1、HDAC2 and HDAC3 either prior to or while these synapse-associated proteins, including PSD-95, SYP and spinophilin, began to be changed significantly by increased APP in APPsw cells.
The number and morphological changes of dendritic spines on hippocampal neurons are considered to be the cellular basis of learning and memory. Spinophilin is a multifunctional protein located on the dendritic spine, and it regulates the membrane and cytoskeleton and plays an important role in the central nervous system. It is closely related to the number and morphology of dendritic spines and the formation of synapses[28, 29]. Our study indicated that BG45 significantly increased the expression of spinophilin. The staining of the cytoskeletal protein F-actin demonstrated that neurites were shorter in the APPsw group than the control group, and the expression of F-actin was also significantly decreased. However, after treatment with 15 µM BG45, the neurites was more abundant and longer, and the cytoskeletal damage was repaired. Therefore, we speculate that in the early stage of AD, both synapses and the cytoskeleton are damaged. However, BG45 specifically alleviated synaptic damage by downregulating the expression of HDAC1、HDAC2 and HDAC3, while it played a role in enhancing synaptic plasticity.
It has been pointed out that nucleation dependent polymerization takes place in Aβ deposition. When an Aβ oligomer is produced, it triggers the first nucleation step of Aβ deposition and accelerates deposition. At the later stage of Aβ aggregation, Aβ fibrils play an important role, and the role of the Aβ oligomer gradually decreases[30, 31]. Soluble Aβ was only detected in APP/Ps1 transgenic mice at 2.5 and 3.5 months of age, while senile plaques were detected in the hippocampus of 7-month-old APP/Ps1 mice, as shown by evaluating the specific emission of broad-spectrum blue violet excitation light from amyloid deposits.
Therefore, in the present study, 2 and 6 months of age were selected as the time window of administration. In addition, a group that received a double dose (one dose at 2 months and one dose at 6 months of age) was set up to explore the effects of early and repeated administration. We found that among the treatment groups, the expression of caspase3 and MAP2 in the hippocampus of the 6mBG45 group was significantly higher than that of Tg+(2+6)m BG45 group or even the Tg+2m BG45 group, whereas the expression of MAP2 was lower in the Tg+6m BG45 group; this indicated that the neuroprotective effect of administration at 2-months of age was more pronounced than that of administration at 6-months of age, that is, BG45 might exert an effect on early damage in this AD model. Furthermore, observation of the production of Aβ plaques and the phosphorylation of the tau protein confirmed that the Tg+2m BG45 group and the Tg+(2+6)m BG45 group, showed similar effects that might be better than those of the Tg+6m BG45 group. Studies with a prolonged experimental period should be carried out to further determine the long-term effects of BG45 on these traits.
To verify the results of the in vitro experiments, we examined the mechanism by which BG45 protects hippocampal neurons. First, BG45 significantly inhibited the expression of HDAC2, especially in the Tg+(2+6)m BG45 group. Furthermore, the expression of synaptophysin in the Tg+(2+6)m BG45 group was higher than the Tg+2m BG45 and Tg+6m BG45 groups. In the Tg+6m BG45 group, the expression of spinophilin and PSD-95 was significantly higher than that in the other two groups, while the effect in the Tg+(2+6)m BG45 group on the decreased spinophilin expression was larger than that in the Tg+2m BG45 group. Overall, BG45 ameliorated the damage to synapse-related proteins in early AD in APP/Ps1 transgenic mice. This is bringing the phenotype towards a WT phenotype.
HDAC, a key enzyme in histone deacetylation, is mainly located in the nucleus. It has been found that knockout of the HDAC2 gene increases the expression levels of other synapse-related genes, such as glutamate ion receptor alginate subunit 2 (GRIK2), synaptophysin (SYNPR), sodium voltage-gated channel beta subunit (SCN3B). In addition, some studies used gene chips to identify genetic pathways related to synaptic function that may be activated by HDACIs. In this study, after treatment with HDACIs, the mRNA levels of GRIK2, SCN3B, SYNPR, Grm2 and Grid2IP, most of which are related to the expression of class I HDAC inhibitors, increased significantly compared with those in the Tg group. These results indicate that among the possible gene pathways activated by HDACIs, BG45 upregulated the expression of GRIK2, SYNPR, SCN3B and Grm2 and grid2IP by inhibiting the deacetylation of HDAC2. We found that most of these genes are related to glutamate receptors, and Grm2 is the gene encoding glutamate receptor subunit 2 (GluR2). Among the treatment groups, the mRNA levels of these genes in the Tg+(2+6) mBG45 group were significantly higher than those in the Tg+2m BG45 and Tg+6m BG45 groups. It can be concluded that BG45 can increase the expression of synapse-related genes in the early stage of AD, and the effect was the most obvious in the Tg+(2+6)m BG45 group.
Due to the changes of the synapse-related genes, we focused on the AMPARs which are closely related to synaptic plasticity. AMPARs are ion channel receptors, and composed of four different glutamate receptor subunits (GluRs) 1, 2, 3, and 4. They participate in the regulation of neurotransmitter release, induce and maintain long-term potentiation (LTP) and long-term depression (LTD) events, and participate in the regulation of learning, memory and other activities. Studies have shown that naturally secreted amyloid oligomers can inhibit LTP in the hippocampus in vivo. The absolute magnitude of LTP and LTD is usually compared to assess the deficiency in synaptic plasticity associated with AD. Some researchers found that in 1-month-old transgenic mice, the induction threshold of LTP/LTD showed a tendency to increase LTP at the cost of LTD, while in 6-month-old transgenic mice, this phenotype was reversed to promote LTD and reduce LTP expression. It is concluded that in adult AD mice, the expression of LTP/LTD is altered primarily through synaptic recruitment and phosphorylation of AMPAR, thereby regulating developmental synapse plasticity. During LTP in the hippocampus, GluA1 is first recruited to synapses, and then GluA2 is also recruited to replace GluA1. GluA2/3 interacts with GRIP1/2 and PICK1 through its PDZ domain to form large complexes involved in AMPAR transport. GRIP1 can bind with GRASP, which inhibits the targeting and membrane processes of AMPARs, thus affecting synaptic plasticity.
Impaired function of AMPA receptors is associated with early cognitive impairment in AD. Studies have shown that the levels of Glu receptors 1, 2, and 3 are reduced in the hippocampus of AD patients, leading to a decrease in dendritic spines and loss of NMDA receptors[39, 40]. Related studies have shown that HDACI-mediated improvement of synaptic function may be associated with changes in AMPA receptor expression. In recent years, a study confirmed the adverse effect of Aβ on AMPARs. It was found that in hippocampal neurons, Aβ1−42 oligomers reduced the expression of AMPA receptors on the postsynaptic membrane and reduced the membrane insertion of new AMPARs and the transport and transfer of mitochondria to dendritic spines. Some researchers also found that the Aβ oligomer preferentially affected the AMPARs containing GluA2, which resulted in the loss of AMPARs and dendritic spines on the surface. Moreover, Aβ increased the phosphorylation level of serine 880 (S880) on GluR2 and significantly reduced the number of GluR2 receptors.
Based on these findings, we first detected the expression of the AMPA receptor subunit in an AD cell model. The results showed that the contents of GluA1, GluA2 and GluA3 were decreased in the APPsw cell membrane, while BG45 increased the expression of receptors. In the experiments in APP/Ps1 transgenic mice, it was found that BG45 increased the expression of GluR2/3/4 receptors in transgenic mice, and reduced the phosphorylation level of the serine 880 site (S880) on GluR2. In addition, measurement of gene levels in APP/Ps1 transgenic mice showed that the histone deacetylase inhibitor BG45 blocked the effect of HDACs, activated gene transcription, and upregulated the expression levels of the related genes (GRIK2, SCN3B, SYNPR, Grm2, and Grid2IP); moreover, BG45 blocked the decrease in GluR2 receptor expression in the AD model and reduced the phosphorylation level of GluR2 (S880 site). Therefore, the increased expression of GluR2 binding protein (GRIP1/2) increased the binding of GluR2 to form more transporters, which increased the transport of AMPA receptors; and the inhibitory effect of GluR2 phosphorylation at the S880 site on GluR2 and GRIP1/2 binding was also alleviated by BG45-mediated downregulation of GluR2 phosphorylation. Therefore, we suggest that BG45 may improve synaptic plasiticity by regulating AMPA receptors and changing the expression of synapse-associated proteins.
In conclusion, BG45 reduced the protein expression of APP by specifically inhibiting class I HDACs (HDAC1、HDAC2 and HDAC3), and by decreasing tau phosphorylation, upregulating pre- and postsynaptic protein expression and repairing cytoskeletal damage, BG45 may have improved synaptic plasticity in cell and animal models of early AD. The underlying mechanisms might be associated with the upregulation of AMPARs and synapse-related genes, which further increased the expression of related proteins. This can provide a new idea for the drug treatment of AD. Nevertheless, it is necessary to use neurobehavioral tests to demonstrate the improvement of AD learning and memory function with BG45 treatment. Therefore, in the following studies, we will focus on functional synaptic plasticity in the cells and model animals. And gene chips could be used to identify genetic pathways related to synaptic function that may be activated by HDACIs.